Protein kinase inhibitors

ABSTRACT

Novel isoquinoline derivatives which are useful as inhibitors of protein kinases for experimental, medical, and drug design purposes are disclosed. Preferred compounds which are specific inhibitors of protein kinase B are also disclosed. Furthermore, pharmaceutical compositions comprising these protein kinase inhibitors, and methods of using such compositions for treatment and diagnosis of cancers, diabetes, cardiovascular pathologies, hemorrhagic shock, obesity, inflammatory diseases, diseases of the central nervous system, and autoimmune diseases, are also disclosed.

FIELD OF THE INVENTION

[0001] The present invention relates to isoquinoline derivatives, topharmaceutical compositions containing the isoquinoline derivatives,their use as inhibitors of protein kinase, as well as to processes forthe preparation and use of such molecules.

BACKGROUND OF THE INVENTION

[0002] Protein kinases are involved in the signal transduction pathwayslinking growth factors, hormones and other cell regulation molecules tocell growth, survival and metabolism under both normal and pathologicalconditions. The superfamily of protein kinases includes protein kinase Aand protein kinase C, as well as the more recently discovered proteinkinase B (PKB). PKB is a direct downstream effector ofphosphatidylinositol 3-kinase (PI 3-kinase) and is activated in responseto insulin or growth factors (for review see Kandel and Hay, Exp. CellResearch 253, 210, 1999).

[0003] PKB activation involves phosphorylation of two amino acidresidues, Ser473 and Thr308. PKB is a newly recognized anti-apoptoticprotein kinase whose activity is strongly elevated in humanmalignancies. PKB was originally discovered as a viral oncogene v-Akt inrat T-cell leukemia It was later established that v-Akt is the oncogenicversion of a cellular enzyme PKB/c-Akt, in which a truncated viral groupspecific antigen, gag, is fused in frame to the full length Akt-1 and ismembrane bound whereas PKB/c-Akt is cytoplasmic. Sequencing of Aktrevealed a high degree of homology to PKA (˜75%) and PKC isozymes(˜50%), a fact which lent to its rechristening as PKB.

[0004] The enzyme is activated by the second messenger PIP3 produced byPI′-3-kinase. PIP3 binds to the pleckstrin homology (PH) domains of PKB,recruits it to the membrane where it is phosphorylated and converted toits activated form. Since PKB activation is PI′-3-kinase dependent, thepersistent activation of certain protein tyrosine kinases, such as IGF-1receptor, EGF receptor, PDGF receptor, pp60c-Src, and the like, leads tothe persistent activation of PKB which is indeed encountered in manytumors. Deletions in the gene coding for the tumor suppressor PTEN alsoinduce the persistent activation of PKB/cAkt since it is the negativeregulator of this enzyme. Also, PKB is overexpressed in 15% of ovariancancers, 12% of pancreatic cancers and 3% of breast cancers, and wasshown to produce a survival signal that protects cells from apoptosisthus contributing to resistance to chemotherapy.

[0005] These molecular properties of PKB and its central role intumorigenesis, implies that this protein kinase may be an attractivetarget for novel anti-cancer agents. To date no specific inhibitors ofPKB are known in the art, nor are any of the disclosed inhibitors ofprotein kinases A and C known to act on PKB.

[0006] Hidaka H. et al. (Biochemistry, 32, 5036, 1984) describe a classof isoquinolinesulfonamides having inhibitory activity towards cyclicnucleotide dependent protein kinases (PKA and PKG) and protein kinases C(PKC). The same class of compounds is claimed in EP 061673, whichdiscloses said compounds as having cardiovascular activity. Additionalderivatives of isoquinolinesulfonyl were disclosed by Hidaka in EP109023, U.S. Pat. Nos. 4,456,757, US 4,525,589, and US 4,560,755.

[0007] Antitumor activity has been suggested for some of theseisoquinolinesulfonamides. Martell R. E. et al. (Biochem. Pharm., 37,635, 1988) found effects of two isoquinolinesulfonamides, namely1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7) andN-[2-guanidinoethyl]-5-isoquinolinesulfonamide (HA-1004), which have acertain selectivity for PKC and cyclic nucleotide dependent proteinkinases, respectively, on calcitriol-induced cell differentiation.Further, Nishikawa M. et al., Life Sci., 39, 1101, 1986), demonstratethat the same compound H-7 inhibits cell differentiation induced byphorbol diester.

[0008] International PCT application WO 93/13072 discloses5-isoquinolinesulfonamide derivatives as protein kinase inhibitingagents wherein the claimed compounds all contain two sulfonyl moieties.

[0009] Other classes of compounds known in the prior art (EP-A-397060,DE-A-3914764 and EP-A-384349) showed the capacity of inhibiting proteinkinases, however, said compounds have a chemical structure which istotally different from that of the compounds of the present invention.In addition, international PCT application WO 98/53050 discloses shortpeptides derived from the HJ loop of a serine/threonine kinase whichmodulate the activity of serine/threonine kinases.

[0010] The minimal consensus sequence for efficient phosphorylation byPKB was found by Alessi et al. (Fed. Eur. Biochem. Soc., 399, 333,1996). This is a 7-mer motif with the most active peptide substratehaving the sequence Arg-Pro-Arg-Thr-Ser-Ser-Phe. Internationalapplication WO 97/22360 discloses certain PKB substrate peptides having7-amino acids length, useful as substrate for measuring PKB activity.

[0011] Obata et al. (J. Biol. Chem., 17, 36108, 2000) described the useof an oriented peptide library approach to determine optimal amino acidsequence of the PKB substrate. All the substrates identified containedthe known motif having the sequence Arg-Xaa-Arg-Xaa-Saa-Ser/Thr.

SUMMARY OF THE INVENTION

[0012] It is an object of the present invention to provide smallmolecules useful as inhibitors of protein kinases for medical,therapeutic and drug design purposes. It is yet another object of thepresent invention to provide such molecules which are selectiveinhibitors of protein kinase B.

[0013] One aspect of the present invention involves the preparation ofnovel compounds which inhibit the activity of protein kinases. It hasnow surprisingly been found that certain novel derivatives ofisoquinolinesulfonamides, are protein kinase inhibiting agents, whichproved to be selectively active towards a specific type of proteinkinase, namely protein kinase B.

[0014] Another aspect of the present invention is directed topharmaceutical compositions comprising as an active ingredient at leastone novel isoquinoline inhibitor of protein kinase and to methods forthe preparation of pharmaceutical compositions comprising suchinhibitors of protein kinases.

[0015] Another aspect of the present invention is directed to the use ofpharmaceutical compositions comprising these protein kinase inhibitorsfor production of medicaments useful for the treatment or diagnosis ofdiseases and disorders. The present invention discloses methods oftreatment of disorders wherein protein kinase is involved including butnot limited to cancers, cardiovascular pathologies, hemorrhagic shock,obesity, inflammatory diseases, diseases of the central nervous system,and autoimmune diseases.

[0016] It is another object of the present invention to provide methodsfor modulating the activity of protein kinases in a subject, comprisingadministering a therapeutically effective amount of protein kinaseinhibitors.

[0017] Further objects of the present invention are directed to methodsfor the diagnosis of diseases including in-vitro diagnosis using thecompounds of the present invention, and in-vivo diagnosis involvingadministering a pharmaceutical composition comprising a diagnosticallyuseful amount of a protein kinase inhibitor prepared according to theprinciples of the present invention.

[0018] It is yet another object of the present invention to providesmall molecules that mimic the ATP molecule that binds to the PKB whichare further conjugated to a peptide substrate or peptido-mimeticsubstrate of PKB. Such chimeric compounds according to the inventionpreferably serve as PKB inhibitors with improved activity andselectivity.

[0019] A preferred embodiment according to the present invention has thegeneral formula I:

[0020] wherein:

[0021] R₁-R₆ are each independently selected from the group consistingof hydrogen, a lower alkyl group, a lower alkoxy group, a phenyl group,a lower alkyl substituted with at least one substituent selected fromthe group consisting of a phenyl group, a halogen, hydroxyl, thiol,nitro, cyano, or amino group;

[0022] Y is selected from the group consisting of sulfonyl, carbonyl,carbamate or carbamoyl;

[0023] R₇ is selected from the group consisting of hydrogen, a loweralkyl group, a lower alkoxy group, a phenyl group, a lower alkylsubstituted with at least one substituent selected from the groupconsisting of a phenyl group, a halogen, hydroxyl, thiol, nitro, cyano,or amino group;

[0024] n is 1-2;

[0025] Z₁ and Z₂ are each independently hydrogen or a lower alkyl group;

[0026] R₈ and R₉ are each independently selected from the groupconsisting of a substituted or unsubstituted phenyl, alkylaryl,naphthyl, quinolyl or a halogen, with the proviso that at least one ofR₈ and R₉ is aromatic.

[0027] One currently more preferred embodiment according to the presentinvention is the compound of Formula II:

[0028] wherein R₁ and R₂ are independently selected from the groupconsisting of substituted or unsubstituted phenyl, alkylaryl, naphthyl,quinolyl, or a halogen, with the proviso that at least one of R₁ and R₂is aromatic.

[0029] Another currently more preferred embodiment of the presentinvention comprises a compound of Formula III:

[0030] wherein R₁ is selected from the group consisting of substitutedor unsubstituted phenyl, alkylaryl, naphthyl, quinolyl, or a halogen;and

[0031] R₃ is selected from the group consisting of hydrogen, a loweralkyl group, a lower alkoxy group, substituted or unsubstituted phenylgroup, a lower alkyl substituted with at least one substituent selectedfrom the group consisting of a phenyl group, a halogen, hydroxyl, thiol,nitro, cyano, or amino group.

[0032] One currently most preferred embodiment of the present inventionis the compoundN1-(8-sulfonamide-5-isoquinoline)-N2-(3,3-diphenyl-2-propenyl)-ethylenediamine,denoted hereinbelow as B-11-I of Formula IV:

[0033] An additional most preferred embodiment of the present inventioncomprises a compound of Formula V:

[0034] wherein:

[0035] X is selected from the group consisting of SO₂—NH, S and O;

[0036] Y represents substituted or unsubstituted alkylene of 1-4 carbonsatoms;

[0037] R₁ and R₂ are independently selected from the group consisting ofhydrogen, a lower alkyl group, a lower alkoxy group, substituted orunsubstituted phenyl group, a lower alkyl substituted with at least onesubstituent selected from the group consisting of a phenyl group, ahalogen, hydroxyl, thiol, nitro, cyano, or amino group;

[0038] Z and W at each occurrence are independently selected from thegroup consisting of hydrogen, a halogen, CF₃, a lower alkoxy, OPhe,alkyl, substituted alkyl, phenyl or substituted phenyl;

[0039] m and n are each independently 0-4;

[0040] or, Z and W are connected via a bridge comprising 0-4 carbonatoms connected covalently through single or double bonds.

[0041] It is understood that if m or n is greater than 1 eachsubstituent may be the same or different.

[0042] An example of a currently more preferred compound according toformula V is a compound of formula VI:

[0043] wherein:

[0044] X is selected from the group consisting of SO₂—NH, S and O;

[0045] Y represents substituted or unsubstituted alkylene of 14 carbonsatoms;

[0046] R₁ and R₂ are independently selected from the group consisting ofhydrogen, a lower alkyl group, a lower alkoxy group, substituted orunsubstituted phenyl group, a lower alkyl substituted with at least onesubstituent selected from the group consisting of a phenyl group, ahalogen, hydroxyl, thiol, nitro, cyano, or amino group.

[0047] Essentially all of the uses known or envisioned in the prior artfor protein kinase inhibitors, can be accomplished with the molecules ofthe present invention. These uses include therapy and diagnostictechniques.

[0048] By way of exemplification, the compounds disclosed in the presentinvention were selected for inhibition of Protein kinase B. Using thepreparations and methods disclosed herein it is possible to obtaincompounds which inhibit the activity of other types of protein kinases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049]FIG. 1. The known molecule H-89 and the various diversity regionsin its structure. A is the core, B is the bridge, C is the tail and D isthe moiety connecting A and B.

[0050]FIG. 2. Miniseries of H-89 analogs optimizing the chemistry of thebond connecting regions B and C. An amine and an amide bond weresynthesized and compared, for three different bicyclic A cores and threedifferent C regions

[0051]FIG. 3. Structure Activity Relation (SAR) of purified compoundswith modified C section.

[0052]FIG. 4. SAR of the effect of an additional substitution near thearomatic residue of region C.

[0053]FIG. 5. The effects of compounds B-11-1 (A) and B-11-2 (B) onOVCAR3 cell apoptosis determined by staining with annexin.

DETAILED DESCRIPTION OF THE INVENTION

[0054] It is now disclosed that small molecules according to the presentinvention are inhibitors of protein kinases. In general, it has now beendiscovered that the active molecules according to the present inventionshare certain structural motifs, which may be construed as an “ATPmimetic” motif. This motif, in the broadest terms may be defined ascomprising four distinct regions, which are defined by their functionaland chemical attributes, as will be exemplified hereinbelow.

[0055] The disclosed protein kinase inhibitors are small molecules whichexhibit enhanced specificity toward certain protein kinase subtypes. Inprinciple, the present invention provides for the first time selectiveinhibitors of protein kinase B. The preferred molecules generally have amolecular weight of less than about 1000 daltons. These and furtheradvantages over the background art will become apparent from thedescription of the currently preferred embodiments of the presentinvention.

[0056] The utility of the compositions according to the invention can beestablished by means of various assays as are well known in the art. Thepreferred compounds of the present invention were found to be active ina panel of in-vitro assays, in inhibiting the activity of proteinkinases and in induction of apoptosis in cancer cells.

[0057] Pharmaceutical compositions according to the present inventioncomprising pharmacologically active protein kinase inhibitors and apharmaceutically acceptable carrier or diluent represent anotherembodiment of the invention, as do the methods for the treatment of amammal in need thereof with a pharmaceutical composition comprising aneffective amount of a protein kinase inhibitor according to theinvention. Methods of treatment using the compositions of the inventionare useful for therapy of cancers, diabetes, cardiovascular pathologies,hemorrhagic shock, obesity, inflammatory diseases, diseases of thecentral nervous system, and autoimmune diseases using such compositions.The pharmaceutical compositions according to the present inventionadvantageously comprise at least one protein kinase inhibitor. Thesepharmaceutical compositions may be administered by any suitable route ofadministration, including topically or systemically. Preferred modes ofadministration include but are not limited to parenteral routes such asintravenous and intramuscular injections, as well as via nasal or oralingestion.

[0058] In the specification and in the claims the term “therapeuticallyeffective amount” refers to the amount of protein kinase inhibitor orcomposition comprising same to administer to a host to achieve thedesired results for the indications described herein, such as but notlimited of cancers, diabetes, cardiovascular pathologies, hemorrhagicshock, obesity, inflammatory diseases, diseases of the central nervoussystem, and autoimmune diseases.

[0059] In the specification and in the claims the term “protein kinase”refers to a member of an enzyme superfamily which functions tophosphorylate one or more protein as described above.

[0060] As used herein and in the claims, the term “inhibitor” isinterchangeably used to denote “antagonist” these terms definecompositions which have the capability of decreasing certain enzymeactivity or competing with the activity or function of a substrate ofsaid enzyme.

[0061] Certain abbreviations are used herein to describe this inventionand the manner of making and using it. For instance, ATP refers toadenosine three phosphate, BSA refers to bovine serum albumin, BTCrefers to bis-(trichloromethyl)carbonate or triphosgene, DCM refers todichloromethane, DIEA refers to diisopropyl-ethyl amine, DMF refers todimethyl formamide, EDT refers to ethanedithiol, EDTA refers to ethylenediamine tetra acetate, ELISA refers to enzyme linked immuno sorbentassay, EGF refers to epithelial growth factor, FACS refers tofluorescence assisted cell sorter, HA refers to hemagglutinin, HBTUrefers to 1-hydroxybenztriazolyltetramethyl-uronium, HEPES refers to4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HOBT refers to1-hydroxybenzotriazole, HRP refers to horse raddish peroxidase, IGFrefers to insulin growth factor, MOPS refers to4-morpholinepropanesulfonic acid, MPS refers to multiple parallelsynthesis, NMP refers to N-methyl formamide, OPD refers too-Phenylenediamine, PBS refers to phosphate buffer saline, PKA refers toprotein kinase A, PKB refers to protein kinase B, PKC refers to proteinkinase C, rpm refers to rounds per minute, SAR refers tostructure-activity relationship, THF refers to tetrahydrofuran, TISrefers to tri-isopropyl-silane, TFA refers to trifluoric acetic acid.

[0062] Pharmacology

[0063] The compounds of the present invention can be administered to asubject in a number of ways, which are well known in the art.Hereinafter, the term “subject” refers to the human or lower animal towhom compounds of the present invention are administered.

[0064] The novel pharmaceutical compositions of the present inventioncontain in addition to the active ingredient conventionalpharmaceutically acceptable carriers, diluents and the like. Solidcompositions for oral administration such as tablets, pills, capsules orthe like may be prepared by mixing the active ingredient withconventional, pharmaceutically acceptable ingredients such as cornstarch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, dicalcium phosphate and gums with pharmaceutically acceptablediluents. The tablets or pills can be coated or otherwise compoundedwith pharmaceutically acceptable materials known in the art to provide adosage form affording prolonged action or sustained release. Other solidcompositions can be prepared as suppositories, for rectaladministration. Liquid forms may be prepared for oral administration orfor injection, the term including subcutaneous, transdermal,intravenous, intrathecal, and other parenteral routes of administration.The liquid compositions include aqueous solutions, with or withoutorganic cosolvents, aqueous or oil suspensions, flavored emulsions withedible oils, as well as elixirs and similar pharmaceutical vehicles. Inaddition, the compositions of the present invention may be formed asaerosols, for intranasal and like administration. More preferredformulations include sustained release or depot formulations which mayprovide a steady state pharmacokinetic profile.

[0065] However, it is evident to the man skilled in the art that dosageswould be determined by the attending physician, according to the diseaseto be treated, method of administration, patient's age, weight,contraindications and the like.

[0066] The compounds defined above are effective as inhibitors ofprotein kinase and can be used as active ingredients of pharmaceuticalcompositions for treatment of one, or simultaneously several, symptomsof the disorders defined above.

[0067] The compounds of the present invention are administered for theabove defined purposes in conventional pharmaceutical forms, with therequired solvents, diluents, excipients, etc. to produce aphysiologically acceptable formulation. They can be administered by anyof the conventional routes of administration.

[0068] It will be appreciated that the most appropriate administrationof the pharmaceutical compositions of the present invention will dependon the type of disorder or disease being treated.

[0069] Chemistry:

[0070] Known inhibitors of protein kinases were used to perform apreliminary screen for PKB inhibition. Among these known protein kinaseinhibitors, the known PKA inhibitor compound H-89 was unexpectedly foundto inhibit PKB activity with an IC₅₀ of 2.4 μM, while inhibiting PKAactivity with IC₅₀ of 48 nM and PKC activity with IC₅₀ of 31 μM.

[0071] The disclosed compounds of the present invention were identifiedfollowing a structure-activity relationship study involving rational andcombinatorial modification ofN-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide also knownas H-89 (Chijiwa et al. J. Biol. Chem. 265, 5267, 1990) as depicted inFIG. 1.

[0072] Some of the preferred compounds of the present invention mayconveniently be prepared using solution phase synthesis methods. Othermethods known in the art to prepare isoquinoline compounds like those ofthe present invention, can be used and are comprised in the scope of thepresent invention.

[0073] By way of exemplification of the principles of the presentinvention, a search for inhibitory PKB compounds focused on SAR studiesof the H-89 molecule, as exemplified hereinbelow. This was followed bySAR study of additional known protein kinase inhibitors that wereunexpectedly identified as PKB inhibitors.

[0074] A preferred embodiment according to the present invention has thegeneral formula I:

[0075] wherein:

[0076] R₁-R₆ are each independently selected from the group consistingof hydrogen, a lower alkyl group, a lower alkoxy group, a phenyl group,a lower alkyl substituted with at least one substituent selected fromthe group consisting of a phenyl group, a halogen, hydroxyl, thiol,nitro, cyano, or amino group;

[0077] Y is selected from the group consisting of sulfonyl, carbonyl,carbamate or carbamoyl;

[0078] R₇ is selected from the group consisting of hydrogen, a loweralkyl group, a lower alkoxy group, a phenyl group, a lower alkylsubstituted with at least one substituent selected from the groupconsisting of a phenyl group, a halogen, hydroxyl, thiol, nitro, cyano,or amino group;

[0079] n is 1-2;

[0080] Z₁ and Z₂ are each independently hydrogen or a lower alkyl group;

[0081] R₈ and R₉ are each independently selected from the groupconsisting of a substituted or unsubstituted phenyl, alkylaryl,naphthyl, quinolyl or a halogen, with the proviso that at least one ofR₈ and R₉ is aromatic.

[0082] One currently more preferred embodiment according to the presentinvention is the compound of Formula II:

[0083] wherein R₁ and R₂ are independently selected from the groupconsisting of substituted or unsubstituted phenyl, alkylaryl, naphthyl,quinolyl, or a halogen, with the proviso that at least one of R₁ and R₂is aromatic.

[0084] Another currently more preferred embodiment preferred of thepresent invention comprises a compound of Formula III:

[0085] wherein R₁ is selected from the group consisting of substitutedor unsubstituted phenyl, alkylaryl, naphthyl, quinolyl, or a halogen;and

[0086] R₃ is selected from the group consisting of hydrogen, a loweralkyl group, a lower alkoxy group, substituted or unsubstituted phenylgroup, a lower alkyl substituted with at least one substituent selectedfrom the group consisting of a phenyl group, a halogen, hydroxyl, thiol,nitro, cyano, or amino group.

[0087] One currently most preferred embodiment of the present inventionis the compoundN1-(8-sulfonamide-5-isoquinoline)-N2-(3,3-diphenyl-2-propenyl)-ethylenediamine,denoted hereinbelow as B-11-1 of Formula IV:

[0088] The currently most preferred embodiment disclosed in formula IV,inhibits 50% of PKB activity at a concentration of 34 μM (fullinhibition at 20 μM) while its 50% inhibition of PKA activity occurs ata concentration of 9-10 μM. For the sake of comparison, the IC₅₀ valuesfor protein kinase inhibition by the known compound H-89 are 2.4 IM forPKB and 50 nM for PKA.

[0089] There are biological experiments (example 4) indicates that thiscompound is capable of penetrating into cells. This characteristic makesit a preferred candidate for use as therapeutic composition.

[0090] This compound denoted B-11-1 includes an additional phenyl moietyand β substitution on the double bond adjacent to the phenyl moiety ofH-89. These modifications contribute to its specificity.

[0091] The compound B-11-2, which is a N-dialkylated form of B-11-1 doesnot inhibit PKB activity and does not induce apoptosis, suggesting thatthe apoptosis induced by B-11-1 is mediated by PKB inhibition.

[0092] An additional most preferred embodiment of the present inventioncomprises a compound of Formula V:

[0093] wherein:

[0094] X is selected from the group consisting of SO₂—NH, S and O;

[0095] Y represents substituted or unsubstituted alkylene of 1-4 carbonsatoms;

[0096] R₁ and R₂ are independently selected from the group consisting ofhydrogen, a lower alkyl group, a lower alkoxy group, substituted orunsubstituted phenyl group, a lower alkyl substituted with at least onesubstituent selected from the group consisting of a phenyl group, ahalogen, hydroxyl, thiol, nitro, cyano, or amino group;

[0097] Z and W at each occurrence are independently selected from thegroup consisting of hydrogen, a halogen, CF₃, a lower alkoxy, OPhe,alkyl, substituted alkyl, phenyl or substituted phenyl;

[0098] m and n are each independently 0-4;

[0099] or, Z and W are connected via a bridge comprising 04 carbon atomsconnected covalently through single or double bonds.

[0100] It is understood that if m or n is greater than 1 eachsubstituent may be the same or different.

[0101] An example of a currently more preferred compound according toformula V is a compound of formula VI:

[0102] wherein:

[0103] X is selected from the group consisting of SO₂—NH, S and O;

[0104] Y represents substituted or unsubstituted alkylene of 14 carbonsatoms;

[0105] R₁ and R₂ are independently selected from the group consisting ofhydrogen, a lower alkyl group, a lower alkoxy group, substituted orunsubstituted phenyl group, a lower alkyl substituted with at least onesubstituent selected from the group consisting of a phenyl group, ahalogen, hydroxyl, thiol, nitro, cyano, or amino group.

[0106] Protein kinases have more than one active site, as they possess acatalytic site for ATP and a substrate binding site. Additionalpreferred compounds according to the present invention can bind bothsites at the same time and may have a synergistic effect that will giveit unique potency and selectivity properties. These preferred compoundsare chimeric molecules which are designed to include an ATP-mimeticmolecule, connected via various spacers to a substrate-mimetic portion.

[0107] Biological Screening Assays for Inhibition of Protein KinaseActivity:

[0108] Inhibition of Enzyme Activity in Cell Free system: PKA ActivityAssay:

[0109] PKA activity is assayed on a 7-mer peptide, LRRASLG, known askemptide. The assay is carried out in 96-well plates, in a final volumeof 50 μl per well. The reaction mixture includes various concentrationsof the inhibitor, 50 mM MOPS, 10 mM MgAc, 0.2 mg/ml BSA, 10 μM ATP, 20μM Kemptide and 1 μCi γ³²P ATP. Reaction is started with addition of 15μl of the catalytic subunit of PKA diluted in 0.1 mg/ml BSA, 0.4 U/well.Two blank wells without enzyme are included in every assay. The platesare agitated continuously at 30° C. for 10′. Reaction is stopped byaddition of 12 μl 200 mM EDTA. 20 μl aliquots of the assay mixture arespotted onto 2 cm² phosphocellulose strips (e.g. Whatman P81) andimmersed in 75 mM phosphoric acid (10 ml per sample). Thephosphocellulose strips are washed 6 times. Washes are done incontinuous swirling for 5 minutes last wash is in acetone. After airdrying the strips, radiation is measured by scintillation spectrometry.Screening of libraries is done in duplicates with a single concentrationof the inhibitor (5 EM). purified compounds are checked in variousconcentrations and their IC₅₀ value is determined.

[0110] Inhibition of Enzyme Activity in Cell Free System: PKB ActivityAssay:

[0111] PKB activity is assayed as described in Alessi et al. (FEBSLetters 399, 333, 1996) with the following modifications: instead ofHA-PKB coupled to beads, soluble His-HA-PKB is used followingprecipitation on a Nickel column. The enzyme activity measurement isperformed as described in the assay for PKA.

[0112] Assays for Inhibition of PKB Activity in Intact Cells:

[0113] Several cancer cell lines were used to determine the activity ofPKB inhibitors in intact cells. For example OVCAR3 is a cell line ofovarian carcinoma with an amplification of the PKB gene, U87MG is aglioma cell line with a deletion of PTEN gene—causing high activity ofPKB, and PANC1 is a pancreatic carcinoma cell line with an amplificationof PKB gene.

[0114] a. Annexin-V Assay for Apoptosis:

[0115] OVCAR3 cells were seeded in 10 cm plates (2×10⁶ cells/plate) andtreated with different concentrations of the inhibitor. 40 hours aftertreatment cells were trypsinysed, washed twice with PBS and suspended inannexin-V buffer annexin-V (Roche) is diluted 1:250 in a buffercontaining 10 mM HEPES pH 7.4, 140 mM NaCl, 5 mM CaCl₂ and 0.2 nMpropidium iodid (PI). Apoptosis measurement was performed by FACSanalysis.

[0116] b. Growth Inhibition:

[0117] OVCAR3, U87MG and PANC1 cells were seeded in 96-well plates. Ineach plate cells were treated with different concentrations (0, 5, 10,25, 50, 75, 100 μM) of the inhibitor, in triplicates. Every day oneplate was fixed by 0.5% gluterdialdehyde, and the inhibitor was replacedin the rest of the plates. After fixation the cells were stained withmethylene-blue 1% for one hour. Plates were washed with distilled waterand dried. Extraction of color was performed by adding 0.1 M HCl for onehour at 37° C. Quantitation of color intensity was performed bymeasurement of the optical density at 620 nm by ELISA reader.

[0118] c. Inhibition of Phosphorylation:

[0119] Cells were seeded in 6-well plates, and treated with differentconcentrations of the inhibitor. Treatment was taken either under serumcontaining media or under starvation for different time periods. Aftertreatment cells are stimulated for 10′ with IGF-1 (HEK-293 and PANC1cells) or EGF (OVCAR3 and U89MG cells). Cell lysates are prepared usingboiled sample buffer. Western blot analysis with a phospho-GSK3 showeddecrease in GSK3 phosphorylation. The effect was also tested on GSK3phosphorylation by expression of kinase-dead-PKB in 293 cells.

[0120] Transfer ELISA Assay for Measuring PKB Activity and Inhibition.

[0121] The inhibitor tested is dissolved in water to the desiredconcentration. Five μl of the inhibitor solution is added to the wellsof a V shaped polyproplylene microplate. Five μl of substrate peptide(Biotin-Lys-Gly-Arg-Pro-Arg-Thr-Ser-Ser-Phe-Ala-Glu-Gly) solution inwater at a concentration of 300 μM is then added to the wells (finalassay concentration is 100 μM). Then PKB enzyme dissolved in 3× reactionmixture (50 mM Tris HCI pH 7.5, 0.1% beta mercaptoethanol, 1 μM PKI(Calbiochem), 10 mM Mg acetate, ATP 5 μM), is added in pre-calibratedamount to the wells. The amount of enzyme is calibrated so that lessthan 10% of the substrate is phosphorylated by the end of the reactionas evaluated by mass spectral analysis. The plate is covered with anadhesive tape, placed over a 1 mm ID vortex at 30° C. and incubated for30 min to 1 hour as needed. At the end of the incubation period 5 μl of0.5 M disdium EDTA are added to the wells followed by 180 μl of PBS.

[0122] For ELISA, a microplate (Costar A/2) is coated with 20 μl of 10μg/ml of avidin in PBS (over night at 4° C. or 30 minutes at 37° C., ona 1 mm ID vortex). The plate is than washed several times with dionizedwater and flicked dry on a towel paper. The wells are filled with 20 μlof PBT (PBS+1% BSA+0.05% tween 20). Five μl from the enzyme reactionplate are transferred to the ELISA plate. The ELISA plate in placed onthe 1 mm ID vortex and incubated for 10 min at RT. The plate is thanwashed with water as before. To each well 20 μl of anti phosphopeptideantibody (Cell Signaling Technology) diluted 1:1000 in PBT are added.The plate is placed again on the vortex, incubated for 30 minutes andwashed with water as before. To each well 20 μl of goat anti-rabbit Igconjugate with horse raddish peroxidase (HRP) is added. The plate isplaced on the vortex, incubated for 20 min and washed with water asbefore. To each well is added 20 μl of HRP substrate (Sigmafast OPD).After sufficient color development (up to maximum of about 30 minutesdevelopment time) the reaction is terminated by the addition of 20 μlper well of 4 M HCl in water. The plate is than read using an ELISAreader at 490 nm. The signal obtained from wells containing potentialinhibitors is compared to signal obtained from wells containing only theenzyme without inhibitor (maximum signal) and wells not containingenzyme (minimum signal).

[0123] The fraction of phosphorylated peptide can be also analyzed bymass spectra following desalting on a ziptip (C18, Millipore i). Mass ofdouble charged substrate peptide is 759.3 Dalton, and of the doublecharged phosphorylated peptide is 799.3 Dalton.

EXAMPLES Example 1 Screening PKA Inhibitors for PEB Inhibition

[0124] Since there are no known inhibitors of PKB, the structuralsimilarity between PKB and other protein kinases was used to screencommercially available inhibitors of other protein kinases, e.g., PKAand PKC, for PKB inhibition. The preliminary screen was conducted inorder to define some structural motifs in active compounds that wouldassist in the initial design of a combinatorial library of candidatecompounds.

[0125] It should be noted, however, that though this approach is veryuseful for rapid identification of lead molecules, the molecules thatare identified would possess inhibition activity against other kinasesas well. Thus, this approach dictates research directed not only atoptimization of the inhibitory activity, but also, and perhaps mostimportantly, specificity-oriented research. Namely, substantial effortsare actually directed at modifying the selectivity profile, in order toobtain a profile of selectivity or specificity towards PKB.

[0126] The screen yielded two compounds that inhibited PKB in the 2-3 μMrange. H-89, a known PKA inhibitor, was chosen to be the basic scaffoldfor the design of the first library, based on its structure and onsynthetic and specificity considerations.

Example 2 Modification of H-89 for Identification of PKB Inhibitors

[0127] The structure of H-89 makes it an ideal candidate for SAR studyusing combinatorial chemistry, since it allows diversity in many regionsof the molecule. The isoquinoline moiety could be replaced with variousbicyclic and aromatic residues, the ethylenediamine bridge can vary inlength, hydrogen bonding properties and substitution, and the cinnamoylmoiety can be modified to a large variety of structures for theevaluation of the optimal properties of this region. In addition, thesulfonamide group can be replaced with carbonyl and other similarmoieties. FIG. 1 shows the various regions of diversity in the structureof H-89 which were used to construct combinatorial libraries. Thelibraries were designed to explore each region's contribution to theinhibition potency and to the specificity against other kinases.

[0128] Region A. A diversity of bicyclic cores, and aromaticheterocyclic cores.

[0129] Region B. A diversity of spacers modified in length,electrostatic properties and substitutions.

[0130] Region C. A diversity of “tails”, either aliphatic or aromatic,differing in length, electrostatic and steric properties.

[0131] Region D. Replacement of the sulfonamide with a carboxy amide,urea, amine, a simple methylene, etc.

[0132] H-89 analogs for SAR study were synthesized in parallel in96-well format using solution chemistry. The first library studiedregion C and examined the necessity of an aromatic residue in that areaFour bicyclic cores were used in this plate, thus each of the aliphaticmoieties that were studied, was tested with the original 5-isoquinolinecore, and three other alternative bicyclic systems. The four different Acores were coupled each with 16 different B+C modified regions andscreened in-vitro for inhibition of PKA and PKB enzyme activity. Noactivity was observed in this library, including the compoundscontaining 5-isoquinoline moiety and resembling H-89 in all featuresexcept the presence of an aromatic residue. The conclusion was thatinteraction with a highly hydrophobic feature in the C region is veryimportant, and the next libraries were designed accordingly.

[0133] Before designing the next library, a miniseries of 9 compoundsaimed at optimization of the chemistry of the bond connecting regions Band C, was synthesized. An amine bond (as in H-89) and an amide bondwere synthesized and compared, for three different bicyclic A cores andthree different C regions. The compounds are depicted in FIG. 2,together with the IC₅₀ for PKB inhibition.

[0134] The results of the miniseries clearly show that an amide bond isless favorable for the inhibition of PKB than an amine. It also impliesthat either the length of the chain connecting the aromatic residue ofpart C to the amine, or the presence of a double bond in it, is alsosignificant, although the effect seems less pronounced than theconversion of the amine to an amide. Based on these results, we turnedto design the next several libraries. They were all based on an aminebond connecting part C and B, and an aromatic residue in part C.

[0135] The diversity regions examined in the next libraries were thecore A, the length and type of the diamino bridge B, and the tail C.These modifications were studied in three different libraries:

[0136] i. Several cores in region A and a diversity of substitutions atregion C.

[0137] ii. Several diamino bridges at region B and a diversity ofsubstitutions at region C.

[0138] iii. Additional region A cores studied with a limited number ofregion C substitutions.

[0139] All libraries were screened for both PKB and PKA inhibition, toassess the contribution of each structural modification to the activityof either enzyme. As discussed before, since the initial lead is a knownPKA antagonist, the SAR should be directed mainly at identifying thespecific enzyme-ligand interactions, which are not common to both PKAand PKB. Naturally, finding these non-mutual interactions will assist indesigning more potent and selective PKB inhibitors.

[0140] For example, it would be desirable to identify structuralfeatures which are essential for the PKA inhibition but that have littleno effect on PKB inhibition, and use them to improve the desiredselectivity. In other words, these essential structural features may bealtered, thereby decreasing the PKA inhibitory activity withoutadversely affecting the PKB inhibitory activity.

[0141] Analysis of the SAR results clearly shows that at region A, noneof the cores that were tested worked, except of the 5-isoquinoline.Replacement with any other core, either as a sulfonamide or acarboxamide derivative, eliminated activity. On region B, elongation ofthe bridge from two to three carbons diminished activity, as didsubstitution on the chain. Using cyclic secondary amine derivativesresulted in elimination of the activity in the case of homopiperazine,implying that the amino protons are important to the interaction,possibly through hydrogen bonding. Surprisingly, the piperazinederivatives showed activity, but significantly less pronounced than thecorresponding ethylenediamine analogs.

[0142] The most significant SAR was observed in the diversity ofmoieties used for region C. The results show that activities ofcompounds derived from 5-isoquinoline-sulfonamide-ethylenediamine, withvarious moieties at region C, vary from almost no activity to verysignificant activities. Notably, significant variation was observed alsoin the activity of PKA. In several cases, the inhibition of PKA wassignificantly decreased. Several of the active compounds from thelibrary were selected for re-synthesis, purification and fullcharacterization, and IC₅₀ values for inhibition of PKA and PKB weredetermined. The results are compiled in FIG. 3.

[0143] As discussed before, identifying non-mutual interactions wasimportant for the design of specific inhibitors. The SAR resultsdepicted in FIG. 3 indicate that an additional bulky substitution at thecarbon bearing the aromatic residue in region C, is unfavorable for PKAinhibition, but has a negligible effect on PKB inhibition. To verifythis conclusion, we synthesized a series of compounds bearing such abulky substitution, and some representative results are depicted in FIG.4 (the results are represented either as IC₅₀ values or as percentinhibition). The results demonstrate that molecules with additionalhydrophobic substitution next to the aromatic residue in region C showedsignificant decrease in PKA inhibition, and a very small effect on PKBinhibition. These results confirm that this position is indeed a PKA“irritant”, shifting its IC₅₀ value for 50 nM to several μM, while itseffect on PKB is minor. The identification of this feature is crucial tothe further optimization of the disclosed compounds. Since the twoenzymes are so similar, a major potential problem is that anymodification we make that will improve affinity for PKB, will probablyhave the same effect on PKA. However, by having the ability to introducea bulky substitution at the identified position we can selectivelydecrease the activity of PKA with only negligible effects on theactivity of PKB.

Example 3 Detailed Synthesis of the Lead Compound B-11-1

[0144] The following is an example of a method of manufacturing thecompound B-11-1.

[0145] (1) 5-isoguinoline N-(3-aminoethyl)sulfonamide

[0146] One gr. (4.7 mMole) of 5-isoquinoline sulfonic acid weredissolved in 0.5 ml of DMF and added 4.6 ml (13 equivalents) of thionylchloride. The mixture was refluxed for 2 hours, cooled and evaporated todryness. The residue was added very slowly to a pre-cooled (0) flaskcontaining 3.6 ml (10 equivalents) of ethylene diamine in methylenechloride. The reaction was stirred at RT for 6 hrs. workup was done byextraction with Water and chloroform, and the organic layer wasevaporated and chromatographed on silica using 5%-15% gradient ofmethanol in chloroform. Clean product was obtained in 35% yield. MS:252, NMR: 2.64(t, 2) 2.917(t, 2) 7.81(t, 1) 8.38(d, 1) 8.45(d, 1) 8.54(d, 1) 8.61(d, 1) 9.3(s, 1).

[0147] (2)N1-(8-sulfonamide-5-isoquinoline)-N2-(3,3-diphenyl-2-propenyl)-ethylenediamine,Denoted B-11-1

[0148] Twenty five mg of 5-isoquinoline N-(3-aminoethyl)sulfonamide (1)were dissolved in methanol and added 64 mg of NaBCNH₃ and a trace ofacetic acid. (approx. 1%). After 5 minutes of stirring, 19 mgs ofdiphenyl cinnamaldehyde were added, and the reaction was stirred for 3hrs. Workup was dine by addition of basic water (NaHCO₃) and chloroformand extraction. The organic layer was evaporated and chromatographed onsilica with 3% methanol in methylene chloride. The purified product(NL-71-101-b) was obtained in 65% yield.

Example 4 Further Biological Evaluation of B-11-1

[0149] The best compound derived from the exemplary SAR study disclosedhereinabove was B 11-1. This compound exhibits greater selectivitytoward PKB than the original compound H-89, since its potency ininhibiting PKA activity is about 330 times lower. The results of enzymeinhibition in the cells-free assay by both compounds are summarized intable 1. TABLE 1 Inhibition of PKA and PKB enzyme activity (μM). EnzymeCompound inhibited B-11-1 H-89 PKB 3.7 1.4 PKA 8.6 0.026

[0150] B-11-1 was further characterized and tested for inhibition of PKBin additional assays:

[0151] B11-1 potency for inhibition of PKB activity in whole cellsdetermined as induction of apoptosis was measured in the Annexin-Vassay. The results of the effects of are summarized in table 2: TABLE 2Induction of apoptosis by B-11-1 treatment  0 vehicle 25 μM 50 μM 75 μM100 μM % apoptosis 10 12 11 19 42 64

[0152] The compound B-11-2, which is an N-dialkylated form of B-11-1 anddoes not inhibit PKB in-vitro, did not induce apoptosis as described inFIG. 5B, suggesting that the apoptosis induced by B11-1 (FIG. 5A) ismediated by PKB inhibition.

[0153] The activity of B-11-1 was also determined by cells growthinhibition assay in OVCAR3 and U87MG cells. The compound was tested inconcentrations of 0, 5, 10, 25, 50, 75, 100 μM. The results showed thatconcentrations of 75 and 100 μM were lethal for the cells after 48hours. 50 μM were lethal after 72 hours, while 25 μM and lowerconcentration had minimal effects. The two high concentrations also hadlethal effects on PANC1 cells after 48 hours treatment. IC₅₀ for growthinhibition of PANC1 cells was determined after 72 hours treatment. TheIC₅₀ found was around 30 μM.

[0154] B-11-1 exhibits inhibition of PKB activity in intact cells asdetermined by western blot analysis with a phospho-GSK3. The resultsshowed decrease in GSK3 phosphorylation at compound concentration of 100μM. Similar effect on GSK3 phosphorylation was observed by expression ofkinase-dead-PKB in 293 cells.

Example 5 Improvement of B-11-1

[0155] Twenty-four compounds designed for improvement of the activity ofB-11-1, including modifications in region C and D (sulfone substitutedby ether), were synthesized. The structures are presented in the Table3: TABLE 3 Sructure EN-118-105-4

EN-118-105-5e

EN-118-107-1

EN-118-107-2

EN-118-107-7

EN-118-107-8

EN-118-107-9

EN-118-97-2

EN-118-97-4

EN-118-97-5

PTR 6026

PTR 6028

PTR 6032

PTR 6034

PTR 6036

PTR 6038

PTR 6040

PTR 6042

PTR 6044

NL-71-161a

NL-71-159a

EN-118-19

EN-118-29

EN-118-115-4

[0156] The skilled artisan will appreciate that the above examples aremerely illustrative and serve as non limitative exemplification of theprinciples of the present invention and that many variations andmodifications are possible within the scope of the currently claimedinvention as defined by the claims which follow.

1 3 1 7 PRT artificial sequence substrate peptide 1 Arg Pro Arg Thr SerSer Phe 1 5 2 6 PRT artificial sequence substrate peptide 2 Arg Xaa ArgXaa Xaa Xaa 1 5 3 12 PRT artificial sequence substrate peptide 3 Lys GlyArg Pro Arg Thr Ser Ser Phe Ala Glu Gly 1 5 10

What is claimed is:
 1. A compound of Formula I:

wherein: R₁-R₆ are each independently selected from the group consistingof hydrogen, a lower alkyl group, a lower alkoxy group, a phenyl group,a lower alkyl substituted with at least one substituent selected fromthe group consisting of a phenyl group, a halogen, hydroxyl, thiol,nitro, cyano, or amino group; Y is selected from the group consisting ofsulfonyl, carbonyl, carbamate or carbamoyl; R₇ is selected from thegroup consisting of hydrogen, a lower alkyl group, a lower alkoxy group,a phenyl group, a lower alkyl substituted with at least one substituentselected from the group consisting of a phenyl group, a halogen,hydroxyl, thiol, nitro, cyano, or amino group; n is 1-2; Z₁ and Z₂ areeach independently hydrogen or a lower alkyl group; R₈ and R₉ are eachindependently selected from the group consisting of a substituted orunsubstituted phenyl, alkylaryl, naphthyl, quinolyl or a halogen, withthe proviso that at least one of R₈ and R₉ is aromatic.
 2. The compoundof claim 1 wherein R₁-R₆ are hydrogen.
 3. A compound of Formula II:

wherein: R₁ and R₂ are independently selected from the group consistingof substituted or unsubstituted phenyl, alkylaryl, naphthyl, quinolyl,or a halogen, with the proviso that at least one of R₁ and R₂ isaromatic.
 4. The compound of claim 3 wherein R₁ and R₂ are eachindependently an aromatic group.
 5. A compound of Formula III:

wherein R₁ is selected from the group consisting of substituted orunsubstituted phenyl, alkylaryl, naphthyl, quinolyl, or a halogen; andR₃ is selected from the group consisting of hydrogen, a lower alkylgroup, a lower alkoxy group, substituted or unsubstituted phenyl group,a lower alkyl substituted with at least one substituent selected fromthe group consisting of a phenyl group, a halogen, hydroxyl, thiol,nitro, cyano, or amino group.
 6. The compound of claim 5 wherein R₁ isphenyl and R₃ is H.
 7. A compound of Formula V:

wherein: X is selected from the group consisting of SO₂—NH, S and O; Yrepresents substituted or unsubstituted alkylene of 14 carbons atoms; R₁and R₂ are independently selected from the group consisting of hydrogen,a lower alkyl group, a lower alkoxy group, substituted or unsubstitutedphenyl group, a lower alkyl substituted with at least one substituentselected from the group consisting of a phenyl group, a halogen,hydroxyl, thiol, nitro, cyano, or amino group; Z and W at eachoccurrence are independently selected from the group consisting ofhydrogen, a halogen, CF₃, a lower alkoxy, OPhe, alkyl, substitutedalkyl, phenyl or substituted phenyl; m and n are each independently 0-4;or, Z and W are connected via a bridge comprising 0-4 carbon atomsconnected covalently through single or double bonds.
 8. A compound ofFormula VI:

wherein: X is selected from the group consisting of SO₂—NH, S and O; Yrepresents substituted or unsubstituted alkylene of 14 carbons atoms; R₁and R₂ are independently selected from the group consisting of hydrogen,a lower alkyl group, a lower alkoxy group, substituted or unsubstitutedphenyl group, a lower alkyl substituted with at least one substituentselected from the group consisting of a phenyl group, a halogen,hydroxyl, thiol, nitro, cyano, or amino group.
 9. A pharmaceuticalcomposition comprising as an active ingredient a compound of Formula I:

wherein: R₁-R₆ are each independently selected from the group consistingof hydrogen, a lower alkyl group, a lower alkoxy group, a phenyl group,a lower alkyl substituted with at least one substituent selected fromthe group consisting of a phenyl group, a halogen, hydroxyl, thiol,nitro, cyano, or amino group; Y is selected from the group consisting ofsulfonyl, carbonyl, carbamate or carbamoyl; R₇ is selected from thegroup consisting of hydrogen, a lower alkyl group, a lower alkoxy group,a phenyl group, a lower alkyl substituted with at least one substituentselected from the group consisting of a phenyl group, a halogen,hydroxyl, thiol, nitro, cyano, or amino group; n is 1-2; Z₁ and Z₂ areeach independently hydrogen or a lower alkyl group; R₈ and R₉ are eachindependently selected from the group consisting of a substituted orunsubstituted phenyl, alkylaryl, naphthyl, quinolyl or a halogen, withthe proviso that at least one of R₈ and R₉ is aromatic.
 10. Thecomposition of claim 9 wherein R₁-R₆ are hydrogen.
 11. A pharmaceuticalcomposition comprising as an active ingredient a compound of the generalFormula II:

wherein R₁ and R₂ are independently selected from the group consistingof substituted or unsubstituted phenyl, alkylaryl, naphthyl, quinolyl,or a halogen, with the proviso that at least one of R₁ and R₂ isaromatic, and a pharmaceutically acceptable diluent or carrier.
 12. Thecomposition of claim 11 wherein R₁ and R₂ are each independently anaromatic group.
 13. A pharmaceutical composition comprising as an activeingredient a compound of Formula III:

wherein R₁ is selected from the group consisting of substituted orunsubstituted phenyl, alkylaryl, naphthyl, quinolyl, or a halogen; andR₃ is selected from the group consisting of hydrogen, a lower alkylgroup, a lower alkoxy group, substituted or unsubstituted phenyl group,a lower alkyl substituted with at least one substituent selected fromthe group consisting of a phenyl group, a halogen, hydroxyl, thiol,nitro, cyano, or amino group.
 14. The composition of claim 13 wherein R₁is phenyl and R₃ is H.
 15. A pharmaceutical composition comprising as anactive ingredient a compound of the general Formula V:

wherein: X is selected from the group consisting of SO₂—NH, S and O; Yrepresents substituted or unsubstituted alkylene of 14 carbons atoms; R₁and R₂ are independently selected from the group consisting of hydrogen,a lower alkyl group, a lower alkoxy group, substituted or unsubstitutedphenyl group, a lower alkyl substituted with at least one substituentselected from the group consisting of a phenyl group, a halogen,hydroxyl, thiol, nitro, cyano, or amino group; Z and W at eachoccurrence are independently selected from the group consisting ofhydrogen, a halogen, CF₃, a lower alkoxy, OPhe, alkyl, substitutedalkyl, phenyl or substituted phenyl; m and n are each independently 0-4;or, Z and W are connected via a bridge comprising 0-4 carbon atomsconnected covalently through single or double bonds.
 16. Apharmaceutical composition comprising as an active ingredient a compoundof the general Formula VI:

wherein: X is selected from the group consisting of SO₂—NH, S and O; Yrepresents substituted or unsubstituted alkylene of 14 carbons atoms; R₁and R₂ are independently selected from the group consisting of hydrogen,a lower alkyl group, a lower alkoxy group, substituted or unsubstitutedphenyl group, a lower alkyl substituted with at least one substituentselected from the group consisting of a phenyl group, a halogen,hydroxyl, thiol, nitro, cyano, or amino group.
 17. A pharmaceuticalcomposition for inhibiting protein kinase comprising as an activeingredient a compound according to any one of claims 1-8.
 18. A methodof treatment of a disease by inhibition of a protein kinase, comprisingadministering to a patient in need thereof a pharmaceutical compositioncomprising as an active ingredient a therapeutically effective amount ofa compound according to any one of claims 1-8.
 19. A method according toclaim 18 wherein the disease is selected from the group comprisingcancers, diabetes, cardiovascular pathologies, hemorrhagic shock,obesity, inflammatory diseases, diseases of the central nervous system,and autoimmune diseases.
 20. A method of diagnosis of a diseasecomprising administering to a patient in need thereof a pharmaceuticalcomposition comprising as an active ingredient a diagnosticallyeffective amount of a compound according to any one of claims 1-8. 21.Use of a compound according to any one of claims 1-8 for preparation ofa medicament for inhibition of a protein kinase.